1. Field of the Invention
The present invention relates to an immunological test method for diagnosing various diseases, blood typing and the like on the basis of antigen-antibody reactions.
2. Description of the Related Art
A microtiter technique of forming a precipitation image by carrier particles on the bottom surface of a reaction vessel has been conventionally popular as a method of detecting an antigen or antibody present in a specimen sample on the basis of an immunological agglutination reaction.
This microtiter technique, however, poses several problems. One of the problems is that a long period of time is required to form a precipitation image because precipitation of the carrier particles and aggregates thereof relies on gravity alone. How to achieve an agglutination reaction by causing the carrier particles to quickly precipitate is the main object to be achieved in the microtiter technique.
In order to provide a means for solving the problem, a method using a magnetic particle as a carrier and applying an external magnetic field to a reaction vessel to quickly form a precipitation image is disclosed in European Patent Application No. 0351857A2. This method using the magnetic carrier will be described with reference to FIGS. 1 to 3 below.
A microplate having a plurality of wells each having a bottom portion with a semispherical or conical recessed surface normally is used in the microtiter technique. FIG. 1A is a plan view of the microplate when viewed from above, and FIG. 1B is a side view showing the layout of the microplate and magnets when a test according to the microtiter technique is to be performed. As shown in FIGS. 1A and 1B, wells 2 formed in a microplate 1 have semispherical bottom surfaces, respectively. Disc-like magnets 4 are respectively located below the wells 2 and are fixed on a support table 3 such that the flat upper surfaces of the magnets 4 are horizontal.
An antigen or antibody is detected using the microplate having the above arrangement. A predetermined amount of a specimen sample and a predetermined amount of a reagent containing a magnetic carrier are distributed to each well 2, and an agglutination reaction is performed. A substance which specifically binds to an antigen or antibody to be detected (to be also referred to as a target substance hereinafter) is immobilized on the magnetic carrier (as described hereinbelow with respect to Example 1).
FIGS. 2A and 3A are views illustrating the principle of measurement according to the microtiter technique. FIGS. 2B and 3B are views showing precipitation images obtained by the microtiter technique.
In the microtiter technique, when a target substance is present in a specimen sample, a plurality of precipitated particles of the magnetic carrier 12 are bonded through the target substance 13 to form an aggregate, as shown in FIG. 2A. The magnetic carrier 12 constituting the aggregate forms a precipitation image uniformly spread on the well bottom surface, as shown in FIG. 2B. This image is called a positive (+) image. When no target substance is present in the specimen sample, a non-agglutinated magnetic carrier 12 is precipitated at the center of the bottom surface of each well 11, as shown in FIG. 3A. The precipitated non-agglutinated magnetic carrier 12 forms a precipitation image concentrated like a button at the center of the well, as shown in FIG. 3B. This image is called a negative (-) image. In this manner, the precipitation images formed on the bottom surfaces of the wells are observed with a naked eye or measured by an optical measuring unit to determine the positive or negative image, i.e., to determine whether an antigen or antibody to be detected is present in the specimen sample.
As described above, a magnetic carrier is used and an external magnetic field is applied to the reaction vessel to shorten the measurement time since a long measurement time is one of the problems of the microtiter technique. This prior art, however, cannot solve all the problems posed by the microtiter technique.
First, in a conventional microtiter technique, factors such as the size and definiteness of a precipitation image depend on the shape of the bottom of a reaction vessel such as a microplate. The shapes and surface areas of the bottoms of reaction vessels are often different in accordance with different manufacturers and different manufacturing lot numbers. Thus, the above factors tend to cause variations between the individual reaction vessels. When the shapes and surface areas of the bottoms of the reaction vessels associated with their volumes are different from each other, compositions, distribution amounts, carrier concentrations, and the like of samples and reagents must be changed to hold a reactivity of the system. For this reason, it is difficult to compare the determination results.
In a conventional microtiter technique, a precipitation image is formed by utilizing the inclination of the bottom surface of a reaction vessel. For this reason, the formed precipitation image tends to be broken by the weight of carrier particles, an electric repulsion force, or external forces such as a magnetic force, a centrifugal force, and a vibration. The degrees of collapse of precipitation images vary depending on the shapes of the bottoms of reaction vessels. Collapse of the precipitation image may cause a determination error such that a specimen sample which must be determined to be positive is determined to be negative, thus posing a decisive problem. Further, in the case where the average grain diameter of carrier particles is 3 .mu.m or less, the formed precipitation image also tends to be broken due to Brownian movement of the particles.
In order to solve this problem, a precipitation image may be formed while a reaction vessel having a flat bottom surface is inclined from the horizontal direction, and the bottom surface of the reaction vessel is then reset to be horizontal. However, when the reaction vessel is moved, the liquid inside the reaction vessel is also moved or vibrated to cause carrier particles constituting the precipitation image to float again or move. Therefore, this method also has poor reliability.